Molecular engineering of anti-CEA (carcinoembryonic antigen) antibodies will be undertaken in a effort to increase their utility in radioimmunotherapy and radioimmunoimaging of adenocarcinomas. Three critical areas in which protein engineering can improve the overall quality and performance of radiolabeled antibodies will be investigated. First, despite the reduction in immunogenicity achieved by production of chimeric T84.66 during the previous project period, in clinical therapy trials a substantial fraction of patients have demonstrated human anti- chimeric antibodies (HACA). Thus, further humanization of the antibody variable regions is of high priority. T84.66 will be humanized using a resurfacing approach: the interior of the Fy will be retained and selected surface residues will be mutagenized to matched conserved surface residues in human antibodies. Thus approach has a high likelihood of retaining the high affinity and specificity of the parental antibody, while reduced immunogenicity will allow administration of multiple doses in clinical trials. Secondly, development of site-specific conjugation of antibodies will allow precise control over the location and extent of radiolabeling, resulting in reproducible production of radioimmunoconjugates with high immunoreactivity and low immunogenicity. Unique cysteine residues (providing reactive thiol groups) or N-linked glycosylation sites (to provide reactive aldehyde groups following periodate oxidation) will be introduced into the CH3 domain of a VL-V H - CH3 "minibody" previously shown to demonstrate excellent xenograft targeting. these minibodies will be conjugated using novel bifunctional chelating agents developed in project 3. Thirdly, fusion to bioactive peptides or antigen-binding domains will be investigated as a means to increase tumor accretion of radiolabeled antibodies. These will include: an anti-CEA-gamma-IFN fusion to locally increase CEA antigen expression in the tumor; an anti-CEA-IL-2 fusion to locally increase vascular permeability at the tumor site; and a bispecific anti-CEA-anti-TAG72 antibody fragment to increase overall uptake in tumors with heterogeneous expression of both antigens. Preadministration of the antibody-cytokine fusions should enhance tumor uptake of subsequently administered radiolabeled antibodies. Tumor targeting by the bispecific fragment will be compared with the corresponding monospecific agent. This combined multidisciplinary approach should yield radioimmunoconjugates with greatly improved efficacy.